Functional imaging of a model unicell: Spironucleus vortens as an anaerobic but aerotolerant flagellated protist.

Advances in optical microscopy are continually narrowing the chasm in our appreciation of biological organization between the molecular and cellular levels, but many practical problems are still limiting. Observation is always limited by the rapid dynamics of ultrastructural modifications of intracellular components, and often by cell motility: imaging of the unicellular protist parasite of ornamental fish, Spironucleus vortens, has proved challenging. Autofluorescence of nicotinamide nucleotides and flavins in the 400-580 nm region of the visible spectrum, is the most useful indicator of cellular redox state and hence vitality. Fluorophores emitting in the red or near-infrared (i.e., phosphors) are less damaging and more penetrative than many routinely employed fluors. Mountants containing free radical scavengers minimize fluorophore photobleaching. Two-photon excitation provides a small focal spot, increased penetration, minimizes photon scattering and enables extended observations. Use of quantum dots clarifies the competition between endosomal uptake and exosomal extrusion. Rapid motility (161 µm/s) of the organism makes high resolution of ultrastructure difficult even at high scan speeds. Use of voltage-sensitive dyes determining transmembrane potentials of plasma membrane and hydrogenosomes (modified mitochondria) is also hindered by intracellular motion and controlled anesthesia perturbs membrane organization. Specificity of luminophore binding is always questionable; e.g. cationic lipophilic species widely used to measure membrane potentials also enter membrane-bounded neutral lipid droplet-filled organelles. This appears to be the case in S. vortens, where Coherent Anti-Stokes Raman Scattering (CARS) micro-spectroscopy unequivocally images the latter and simultaneous provides spectral identification at 2840 cm-1. Secondary Harmonic Generation highlights the highly ordered structure of the flagella.

Mitochondria-derived organelles in the diplomonad fish parasite Spironucleus vortens.

In some eukaryotes, mitochondria have become modified during evolution to yield derived organelles (MDOs) of a similar size (hydrogenosomes), or extremely reduced to produce tiny cellular vesicles (mitosomes). The current study provides evidence for the presence of MDOs in the highly infectious fish pathogen Spironucleus vortens, an organism that produces H2 and is shown here to have no detectable cytochromes. Transmission electron microscopy (TEM) reveals that S. vortens trophozoites contain electron-dense, membranous structures sometimes with an electron-dense core (200 nm-1 µm), resembling the hydrogenosomes previously described in other protists from habitats deficient in O2. Confocal microscopy establishes that these organelles exhibit autofluorescence emission spectra similar to flavoprotein constituents previously described for mitochondria and also present in hydrogenosomes. These organelles possess a membrane potential and are labelled by a fluorescently labeled antibody against Fe-hydrogenase from Blastocystis hominis. Heterologous antibodies raised to mitochondrial proteins frataxin and Isu1, also exhibit a discrete punctate pattern of localization in S. vortens; however these labelled structures are distinctly smaller (90-150 nm) than hydrogenosomes as observed previously in other organisms. TEM confirms the presence of double-membrane bounded organelles of this smaller size. In addition, strong background immunostaining occurs in the cytosol for frataxin and Isu1, and labelling by anti-ferredoxin antibody is generally distributed and not specifically localized except for at the anterior polar region. This suggests that some of the functions traditionally attributed to such MDOs may also occur elsewhere. The specialized parasitic life-style of S. vortens may necessitate more complex intracellular compartmentation of redox reactions than previously recognized. Control of infection requires biochemical characterization of redox-related organelles.

Carbohydrate and amino acid metabolism of Spironucleus vortens.

The metabolism of Spironucleus vortens, a parasitic, diplomonad flagellate related to Giardia intestinalis, was investigated using a combination of membrane inlet mass spectrometry, (1)H NMR, (13)C NMR, bioscreen continuous growth monitoring, and ion exchange chromatography. The products of glucose-fuelled and endogenous metabolism were identified by (1)H NMR and (13)C NMR as ethanol, acetate, alanine and lactate. Mass spectrometric monitoring of gas metabolism in buffered cell suspensions showed that glucose and ethanol could be used by S. vortens as energy-generating substrates, but bioscreen automated monitoring of growth in culture medium, as well as NMR analyses, suggested that neither of these compounds are the substrates of choice for this organism. Ion-exchange chromatographic analyses of free amino-acid and amino-acid hydrolysate of growth medium revealed that, despite the availability of large pools of free amino-acids in the medium, S. vortens hydrolysed large amounts of proteins during growth. The organism produced alanine and aspartate, and utilised lysine, arginine, leucine, cysteine and urea. However, mass spectrometric and bioscreen investigations showed that addition of the utilised amino acids to diluted culture medium did not induce any significant increase in metabolic or growth rates. Moreover, as no significant amounts of ornithine were produced, and addition of arginine under aerobic conditions did not generate NO production, there was no evidence of the presence of an energy-generating, arginine dihydrolase pathway in S. vortens under in vitro conditions.

A 'Sleeping Trojan Horse' which transports metal ions into cells, localises in nucleoli, and has potential for bimodal fluorescence/PET imaging.

A rhenium polypyridine-based molecular vessel is membrane impermeant when empty, but, upon loading with metal ions, the cationic form is taken up by MCF-7 cells, localising in nucleoli. The luminescence of the vessel and its copper binding ability suggest potential as a bimodal fluorescence/PET imaging agent.

Effect of garlic and allium-derived products on the growth and metabolism of Spironucleus vortens.

Spironucleus is a genus of small, flagellated parasites, many of which can infect a wide range of vertebrates and are a significant problem in aquaculture. Following the ban on the use of metronidazole in food fish due to toxicity problems, no satisfactory chemotherapies for the treatment of spironucleosis are currently available. Using membrane inlet mass spectrometry and automated optical density monitoring of growth, we investigated in vitro the effect of Allium sativum (garlic), a herbal remedy known for its antimicrobial properties, on the growth and metabolism of Spironucleus vortens, a parasite of tropical fish and putative agent of hole-in-the-head disease. The allium-derived thiosulfinate compounds allicin and ajoene, as well as an ajoene-free mixture of thiosulfinates and vinyl-dithiins were also tested. Whole, freeze-dried garlic and allium-derived compounds had an inhibitory effect on gas metabolism, exponential growth rate and final growth yield of S. vortens in Keister's modified, TY-I-S33 culture medium. Of all the allium-derived compounds tested, the ajoene-free mixture of dithiins and thiosulfinates was the most effective with a minimum inhibitory concentration (MIC) of 107 µg ml(-1) and an inhibitory concentration at 50% (IC(50%)) of 58 µg ml(-1). It was followed by ajoene (MIC = 83 µg ml(-1), IC(50%) = 56 µg ml(-1)) and raw garlic (MIC >20 mg ml(-1), IC(50%) = 7.9 mg ml(-1)); allicin being significantly less potent with an MIC and IC(50%) above 160 µg ml(-1). All these concentrations are much higher than those reported to be required for the inhibition of most bacteria, protozoa and fungi previously investigated, indicating an unusual level of tolerance for allium-derived products in S. vortens. However, chemically synthesized derivatives of garlic constituents might prove a useful avenue for future research.

The diplomonad fish parasite Spironucleus vortens produces hydrogen.

The diplomonad fish parasite Spironucleus vortens causes major problems in aquaculture of ornamental fish, resulting in severe economic losses in the fish farming industry. The strain of S. vortens studied here was isolated from an angelfish and grown in Keister's modified TY-I-S33 medium. A membrane-inlet mass spectrometer was employed to monitor, in a closed system, O(2), CO(2), and H(2) When introduced into air-saturated buffer, S. vortens rapidly consumed O(2) at the average rate of 62+/-4 nmol/min/10(7) cells and CO(2) was produced at 75+/-11 nmol/min/10(7) cells. Hydrogen production began under microaerophilic conditions ([O(2)]=33.+/-15 microM) at a rate of 77+/-7 nmol/min/10(7) cells. Hydrogen production was inhibited by 62% immediately after adding 150 microM KCN to the reaction vessel, and by 50% at 0.24 microM CO, suggesting that an Fe-only hydrogenase is responsible for H(2) production. Metronidazole (1 mM) inhibited H(2) production by 50%, while CO(2) production was not affected. This suggests that metronidazole may be reduced by an enzyme of the H(2) pathway, thus competing for electrons with H(+).

Uptake and localisation of rhenium fac-tricarbonyl polypyridyls in fluorescent cell imaging experiments.

The synthesis of a series of rhenium fac tricarbonyl bisimine complexes and their application as lumophores in fluorescence imaging of yeast and human adenocarcinoma cells is reported. A wide range of complexes are synthesised with varying charges and lipophilicities, all of which have photophysical properties which make them suitable as cell imaging agents. After attempts to apply these as imaging agents in various strains of yeast which showed limited uptake, an investigation was undertaken of their applications as imaging agents in mammalian cells. In general the uptake was high and short-term toxicity and photobleaching appear to be low. The patterns of uptake and localisation are correlated with structural and electronic features of the complexes in an attempt to establish ground-rules for the design and application of rhenium complexes in imaging of eukaryotes.

Probing intracellular oxygen by quenched phosphorescence lifetimes of nanoparticles containing polyacrylamide-embedded [Ru(dpp(SO3Na)2)3]Cl2.

Methods for measuring O(2) within living cells that rely on luminescent probes are hampered by several factors: local conditions of hydrophobicity, pH, ionic composition, dielectric constant, and photobleaching by free radical species. Use of a polymer-embedded luminophore should minimize these problems. Here we use a Ru(II) coordination complex embedded within 45 nm hydrodynamic diameter nanoparticles, and demonstrate that both phosphorescence intensity and lifetimes are O(2)-sensitive, both in aqueous suspensions and intracellularly (e.g. 4.06 versus 1.55 microseconds under anaerobic or aerobic conditions, respectively). Electroporation is necessary for incorporation of the nanoparticles into yeasts: it is more effective with the fission yeast, Schizosaccharomyces pombe, than for the budding yeast, Saccharomyces cerevisiae. However, electroporation was not required for particle uptake into a cultured human cell-line (mammary adenosarcoma MCF-7), although the intracellular distribution of the probe is more general to intracellular compartments when electroporation is employed. These procedures did not compromise vitality of cells over periods of 6 h, as judged by retention of structural characteristics evident in Nomarski interference or confocal microscopy images. Spatial resolution of intracellular structures defined by nanoparticle phosphorescence intensity imaging indicates potential usefulness of the application of lifetime imaging techniques for mapping of intracellular O(2) distributions.